Use of hyaluronidase for treatment of muscle stiffness

ABSTRACT

Provided are methods and kits for reducing the severity of muscle stiffness. The method comprises delivering to one or more specific locations in the deep fascia of an affected muscle a composition comprising a therapeutically effective amount of hyaluronidase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional patent applicationNo. 62/025,257, filed on Jul. 16, 2014, and U.S. provisional patentapplication No. 62/118,707, filed on Feb. 20, 2015, the disclosures ofwhich are incorporated herein by reference.

BACKGROUND OF THE DISCLOSURE

Muscle stiffness is a common symptom for which no specific etiology hasbeen determined and no treatment exists as of yet. Muscle stiffnessoften occurs from lack of movement, for example after prolonged bed rest(Clavet et al., 2008, CMAJ, March 11, 178(6):691-7), in the elderly(Trindade et al., 2012, J Biomech., January 3, 45(1):199-201; Wojtysiak,2013, Folia Biol (Krakow), 61(3-4):221-6, PubMed PMID: 24279172), withparalysis of limbs due to neurological or muscular diseases, due tometabolic conditions such as diabetes (Duffin, 2002, Diabet Med.December, 19(12):1009-13, PubMed PMID: 12647842), and after excessiveexercises such as running a marathon etc. The severity of musclestiffness can range from an uncomfortable sensation of rigidity toexacerbation of spasticity (if there is a previous central nervoussystem injury). Both rigidity and spasticity may be accompanied bynon-specific pain. The high incidence of muscle stiffness represents anenormous cost to society worldwide.

Spasticity commonly presents as muscle over-activity, reduction in theability to relax specific muscles, hypertonia, paresis, muscle spasms,and loss of fine motor control, attributed to neural mechanisms.However, less understood symptoms of spasticity include increasedstiffness in the soft tissue, muscle fatigue, and postural changes inthe limbs, which can be explained by non-neural/peripheral contributionsthat have secondary effects on skeletal muscles. In fact it has beenshown that spasticity is not an immediate consequence of CNS injury asit progresses during the weeks and months after injury; this suggeststhat there are other non-neural/peripheral factors that contribute tospasticity (Lundstrom et al., 2008, Eur J Neurol., 15(6):533-9).

Surgical, pharmacological, and physiotherapy techniques are among themost common interventions offered to alleviate spasticity.Pharmacological agents include oral medication such as benzodiazepines,baclofen, tizanidine hydrochloride, and dantrolene. Diazepam is one ofthe oldest and most commonly used benzodiazepine for treatingspasticity. Botulinum toxin type A is used for focal treatment of overlyspastic muscles, while intrathecal baclofen is commonly used to reducespasticity in individuals with spinal cord injury. However, even thoughcurrent pharmacological agents significantly reduce spasticity, theiruse does not always translate into increased function because of sideeffects including drowsiness and muscle weakness (Nielsen et al., 2007,Acta Physiol (Oxf), February, 189(2):171-80).

SUMMARY OF THE DISCLOSURE

The present disclosure provides methods and kits for reducing musclestiffness. The methods and kits are based on the observation thatinjection of hyaluronidase in the deep fascia region at or near specificsites—termed as the centers of coordination—results in amelioration ofstiffness of muscles. In one aspect, the present disclosure provides amethod for providing relief from stiffness of a muscle in an individualcomprising the steps of delivering in a region of deep fasciasurrounding the muscle at one or more centers of coordination (CCs) orcenters of fusion (CFs) a composition comprising a therapeuticallyeffective amount of hyaluronidase. Alternatively or additionally,hyaluronidase may be delivered to a region of deep fascia surrounding asecond muscle that affects the function of the first muscle (or whosefunction is affected by the function of the first muscle) at or near oneor more centers of coordination or center of fusion associated with thesecond muscle. This results in reducing stiffness of the first muscle.

The present disclosure also provides kits for reducing stiffness ofmuscles. The kits comprise one or more of: combined or separate doses ofhyaluronidase for one or more injections, administrations aids (such assyringes), charts showing centers of coordination or center of fusionwhere hyaluronidase may be injected, instructions for use, and follow-upguidance. Hyaluronidase may be provided as a combined dose (in a singlevial) or as multiple individual doses, and may be provided in aready-to-use form or in a form that can be reconstituted. If provided ina form that can be reconstituted, the kit may also containreconstitution medium.

DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B. Illustration of centers of coordination in the frontand back of a human body. The various symbols represent: empty circles:anterior CCs related to movement in the frontal (or coronal) plane,filled circles: posterior CCs related to movement in the frontal plane,empty triangles: anterior CCs related to movement in the horizontal (ortransverse) plane, filled triangles: posterior CCs related to movementin the horizontal plane, empty diamonds: anterior CCs related tomovement in the sagittal plane, filled diamonds: posterior CCs relatedto movement in the sagittal plane.

FIGS. 2A and 2B. Illustration of centers of fusion in the front and backof a human body. The various symbols represent: ante-lateral direction(circles), retro-lateral direction (diamonds), ante-medio direction(square) and retro-medio direction (triangles).

FIGS. 3A and 3B. Illustration of layers of the skin and muscles at acenter of coordination. The various layers are illustrated. A thin layerof epimysium is present between the deep fascia and the muscle. The deepfascia is shown as three layers of dense connective tissue (comprisingcollagen fiber type I and III) and two layers of loose connective tissue(comprising adipose cells, GAG and hyaluronic acid). In 3A, the layersof the deep fascia are shown in a non-sliding position and in FIG. 3B,the layers of the fascia are shown after sliding relative to each other.

FIG. 4. Illustration of region of a lower limb showing comparison ofdeep fascia layers of a region which has centers of coordination versusa region which does not. The deep fascia layers at a center ofcoordination are slidable with respect to each other, while the deepfascia layers at regions which do not have centers of coordination orcenter of fusion are fused and therefore are not slidable with respectto each other.

FIGS. 5A-5B, 6, 7A-7C, 8A-8C, 9A-9B, 10A-10B, 11A-11B, and 12A-12F.Illustrations of shoulder flexion/extension movements that can be usedto assess restriction of movement due to muscle stiffness.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure provides a method for reducing or treating musclestiffness with or without pain. The method can be used in neurologicpatients who may have spasticity as well as in non-neurologic patientswith non-specific muscle pain related to muscle stiffness. The methodcomprises administering compositions comprising hyaluronidase into thedeep fascia region at specific locations surrounding the affectedmuscle. Alternatively, or additionally, hyaluronidase administrationsmay be carried out at specific locations in deep fascia region thatsurround other muscles that have an effect on the function of, or whosefunction is affected by, the muscle in question. Such deep fascia regionof a second muscle is considered to be in continuity with the deepfascia region surrounding the affected muscle. The affected muscle isalso referred to herein as the “first muscle” or the “muscle inquestion”. The deep fascia may surround the muscles partially orcompletely.

The present method is based on the observation that injection ofhyaluronidase at or near specific sites (centers of coordination (CCs)or centers of fusion (CFs)) results in reducing stiffness. It was alsoobserved that hyaluronidase injection in other areas (not at or near aCC) does not result in measurable reduction of stiffness. In oneembodiment, the disclosure provides a method for providing relief fromstiffness of a first muscle in an individual comprising the steps ofdelivering in a region of deep fascia surrounding the first muscle or ina region of deep fascia surrounding a second muscle that affects (or isaffected by) the function of the first muscle at or near one or morecenters of coordination or centers of fusion associated with the muscle,a composition comprising a therapeutically effective amount ofhyaluronidase. This results in reducing stiffness of the first muscle.

Hyaluronidase is an enzyme known to degrade hyaluronic acid. It has beenapproved as an adjuvant to increase the spread and dispersion of otheradministered drugs. Hyaluronidase is commercially available as Vitrase,Amphadase, Hydase, and Hylenex. These formulations representhyaluronidase from ovine, bovine and human sources.

In one aspect, the present disclosure provide hyaluronidase compositionsfor use in reducing muscle stiffness. The compositions comprisehyaluronidase in pharmaceutically acceptable carriers (includingphysiological buffers) or in dry form (e.g., powdered or lyophilized).The compositions may comprise multiple doses or a single dose foradministration, such as via injection, to an individual for delivery tothe deep fascia region at specific locations in muscles.

In the present disclosure, hyaluronidase is delivered at or near atleast one center of coordination site or at least one center of fusionsite. In one embodiment, hyaluronidase is delivered to the CC sites byinjections. For examples, hyaluronidase is administered by injections in1 to 10 sites (and all integer values therebetween). In variousembodiments, injections may be given at 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10sites. In one embodiment, injections are given at two or more sites. Inone embodiment, injections may be given in at least 10 sites (such asfrom 10-20 sites). For example, to treat muscle stiffness in limbs, 6-10sites can be injected in the limb at various CC points along the limb.

Centers of coordination represent points in the deep fascia or withinmuscles (perimysium, epimysium) where muscular forces converge. A Centerof coordination is a region of convergence of collagen fibresoriginating from groups of motor units which move a segment in onespecific direction in space (Stecco L. 2004, Fascial Manipulation forMusculoskeletal Pain; Piccin; April 2004, incorporated herein byreference). In one embodiment, a second group of points that could beused are the centers of fusions (CFs). The CFs are the converging pointsfor vectors of intermediate muscular fibres of three planes in the spacein the adjacent directions. (Stecco L. 2004, Fascial Manipulation forMusculoskeletal Pain; Piccin; April 2004, page 149, chapter 15) A CCsite is considered to be a focal point in the deep fascia where vectorforces produced by the contraction of monoarticular and biarticularmuscle fibers of one body segment converge during a precise movement.Being a focal point for the convergence of numerous vectors, CCs arecommonly located at a distance from the relative joint component. Stecco(2004) has mapped out the anatomical location of CCs within each bodysegment.

As shown in FIGS. 1A and 1B, 100 CCs have been identified. In FIGS. 2Aand 2B, 105 CFs have been identified. Each CC and CF is shown as havinga unique arbitrary identifier. The Center of Coordinations may begrouped based on affecting muscles that facilitate movement in thefrontal plane (circles), sagittal plane (diamonds) or horizontal plane(triangles). The CCs in the front of the body are shown as emptysymbols, and those in the back of the body are shown as filled symbols(FIGS. 1A and 1B). The CFs may be grouped based on affecting musclesthat facilitate movement in the ante-lateral direction (circles),retro-lateral direction (diamonds), ante-medio direction (square) orretro-medio direction (triangles) (FIGS. 2A and 2B).

The CCs and CFs are distinguishable as having slidable layers in thedeep fascia region. An illustration of two CCs is shown in FIG. 4. Across-section of a region where a CC is present shows deep fascia layerswhich can slide relative to each other (as illustrated in FIGS. 3A and3B). The two CCs in FIG. 4 correspond with the CCs identified as la-cxand la-pv in FIGS. 1A and 1B. Hyaluronic acid is present between thelayers of the fascia and contributes to the ability of the fasciallayers to slide relative to each other. In contrast, a similarcross-section from an area where no CCs are present shows the deepfascia layers are fused so that the layers lack the ability to sliderelative to each other (FIG. 4).

The present disclosure entails the injection of hyaluronidase at or nearthe CC sites or CF sites. Thus, injections can be performed in a regionwithin about a 1 inch diameter around CCs or CFs to relievedensification of the fascia and restore ease of movement. Inembodiments, injections can be performed in a region within about a0.75. 0.5 or 0.25 inch diameter around the CCs or CFs. We have observedthat the amount of hyaluronidase useful in the present method is higherthan what is typically used for adjuvant or dispersion purposes. Forexample, hyaluronidase can be administered at dosages of 25 USP units to300 USP units per injection (and all dosages to the tenth decimal pointthere between). The terms “unit” or “units” as used herein are intendedto be the same as indicated on the commercially available products.

Further details of the commercially available Hylenex are provided inU.S. Pat. No. 7,767,429 incorporated herein by reference. The amount ofhyaluronidase may be present in a suitable volume for injection. Forexample, in one embodiment, the dosage is 37.5 units (0.25 cc) to 300units (2 cc) per site (CC) injected at desired sites, such as at 6-10sites in the limb at various points along the limb at one or morecenters of coordination.

In the method of the present disclosure, the CC sites for injection areselected based on the affected movement. For example, if restriction ofmovement of a joint is observed in the frontal plane, then hyaluronidaseinjections may be carried out at one or more CCs sites identified ascircles in FIGS. 1A and 1B. If movement is restricted in the sagittalplane, then injections can be performed into one or more CC sitesidentified as diamonds (an and re points), and if movement is restrictedin the horizontal plane, then injections may be performed into one ormore CC sites identified as triangles (ir and er points), and ifmovement is restricted in the frontal plane, the injections can beperformed into one or more CC sites identified as circles (me and lapoints). In the same manner, if movement is restricted in theante-lateral and retro-lateral direction, then injections can beperformed into one or more CF sites identified as diamonds (an-la andre-la points), and if movement is restricted in the ante-medio andretro-medio direction, then injections may be performed into one or moreCF sites identified as triangles (an-me and re-me points). In someembodiments, corresponding CCs on the anterior and posterior areselected. Such embodiments have been found to advantageously provide abalancing effect on the fascia. Depending upon the restriction ofmovement, a combination of CCs in one or more of the frontal, sagittal,and horizontal planes may also be used. For example, if movement of thewrist (carpal joint) is restricted in all planes, the CCs to be injectedmay include an-ca, re-ca, ir-ca, er-ca, la-ca and me-ca.

More commonly, the restriction may be in multiple joints along a singleplane. If the elbow joint is exhibiting stiffness in the sagittal plane,then multiple CC sites may be selected in the same plane across multiplejoints. For example, the CC sites injected could include an-sc, an-hu,an-cu, an-ca, re-hu, re-cu, and re-ca. As another example, if anindividual presents stiffness in knee movement (i. e., movement in thesagittal plane), then CC sites an-cx, an-ge, an-ta, re-ge and re-ta maybe injected. As another example, if an individual presents stiffness inneck movement (i.e., movement in the retro-lateral direction), then CFssites an-la-cl, an-la-sc, re-la-cl and re-la-sc may be injected.

The abbreviations used in the figures and in the description are asfollows—an: ante (front), re: retro (back), ir: internal rotation, er:external rotation, me: medial, la: lateral, sc: scapular (shoulder), hu:humerus (upper arm), cu: cubitus (elbow), ca: carpus (wrist), cx: coxa(hip), ge: genu (knee), ta: talus (lower leg), cl: collum (neck), di:digiti (fingers), cp: caput (head), the thorax (trunk), pe: pes (foot),pv: pelvis, lu: lumbi (lumbar).

Based on the CC and CF site map provided herein, it is within thepurview of those skilled in the art to identify which and how many sitesto select for hyaluronidase injection.

It should be noted that the examples herein are non-limiting, and CCsmay be selected based on the presentation of each patient.

It was observed that a one-time injection in specific sites resulted inrelief of muscle stiffness for at least about 1 month and often longerwith facilitation of passive and/or active mobility. In cases ofspasticity the injections may be repeated. Exercise therapy to restorepassive or active movement in normal planes will facilitate prolongedeffect of the injections.

Hyaluronidase may be administered by any means that will deliver theagent to the relevant site. For example, Hyaluronidase may be injectedlocally into the stiff areas of the connective tissue. Injection (suchas using a syringe) into the deep fascia region is within the purview ofthose skilled in the art. Surface anatomy maps indicate the depth of thepoints with respect to the skin surface, and the resistance in thetissue upon injection indicates that the area of stiffness has beenreached: a grabbing of the needle is often felt in areas that areparticularly stiff Hyaluronidase may also be translocated transdermallyusing iontophoresis or sonophoresis in areas where the skin is thin, andthe fascia more superficial. Iontophoresis utilizes small electriccurrents to enhance transport across the skin by mechanisms such aselectrophoretic and electro-osmotic driving forces. Sonophoresis usesultrasound as a physical enhancer for systemic drug delivery, and caneffectively deliver Hyaluronidase alone or in combination with otheragents regardless of their electrical characteristics. It can also becoupled with iontophoresis and microneedling methods to enhance drugdelivery. Sonophoresis can be used at frequencies in the range of 20kHz-16 MHz and intensities up to 14 W/cm² (spatial average pulse averageintensity, I_(SAPA)) to enhance skin permeability. Low frequencysonophoresis (20 kHz<f<100 kHz) may be particularly useful. Sonophoresismay be directed by ultrasound guidance to areas where the connectivetissue shows reduced elasticity.

The hyaluronidase formulations may comprise other ingredients such asnon-active ingredients. For example, the formulations may compriseexcipients such as Azone (1-dodecylazacycloheptan-2-one or laurocapram),DMSO (dimethyl sulphoxide), and/or surfactants. Such excipients mayincrease transdermal drug transport via several mechanisms such asincreased drug solubility in the donor formulation and drug partitioninginto the subcutaneous space because of the solvent properties of thesecompounds. For example, DMSO, a commonly used topical analgesic,anti-inflammatory, and antioxidant, has been used in studies of skeletalmuscle as a selective antioxidant or as a solvent for numerous drugs.Local anesthetic agents such as lidocaine can be combined withhyaluronidase when there is substantial pain. This may facilitatepost-injection stretching for areas that are already slightlycontracted. Sodium Bicarbonate may be used to increase onset time andprolong the action of hyaluronidase. Saline can be used withhyalruonidase to produce a volume effect and facilitate separation ofthe layers of the fascia. In one embodiment, the only enzyme in theadministered composition is hyaluronidase. In one embodiment, the onlyprotein in the composition is hyaluronidase.

In one embodiment, the individual exhibiting muscle stiffness is treatedwith only hyaluronidase. In one embodiment, the individual is nottreated with benzodiazepines, baclofen, tizanidine hydrochloride, anddantrolene, or botulinum toxin type A or B. In one embodiment, anindividual may be treated with other agents such as benzodiazepines,baclofen, tizanidine hydrochloride, dantrolene in addition to thehyaluronidase treatment. Botulinum toxin injections may be given afterthe effect of the hyaluronidase injections has worn off, but not during,to prevent systemic dispersion of the toxin.

Muscle stiffness may be assessed by standard means well known toclinicians and others skilled in the art. For example, the limitation inpain-free passive or active range of motion at a particular joint(Norkin and White 1995, Measurement of Joint Motion: A Guide toGoniometry. Philadelphia, Pa.: FA Davis Co) may be used. Spasticity orthe resistance to passive movement may be assessed using the modifiedAshworth scale (Bohannon, R. and Smith, M. 1987, Physical Therapy,67(2): 206; Brashear et al., 2002, Archives of physical medicine andrehabilitation, 83(10): 1349-1354). Individuals may be asked to performvarious joint movements and their performance may be evaluated. In oneembodiment, their performance may be recorded on video and analysis canbe carried out using commercially available software (such as Dartfishsoftware).

The present method may be used to restore movement in the limbs andenhance limb function by releasing stiffness in the fascia and musclecaused by prolonged immobility secondary to disuse, orthopedic injury,neurologic causes of paralysis such as stroke, traumatic brain injury,multiple sclerosis, spinal cord injury, cerebral palsy or developmentalcauses of contractures, such as specific subtypes of arthrogryposismultiplex congentia, as well as muscle pain and joint stiffness fromnon-neurologic causes such as from prolonged bed rest, post-operativestiffness, myofascial pain and fibromyalgia, over-use, repetitivetrauma, age-related muscle stiffness and muscle-stiffness due todiabetes.

In one embodiment, additional active ingredients, such as pain killers,anesthetics and the like may be used.

In one embodiment, the hyaluronidase injections are used to restorerange of motion without causing further muscle paralysis (which occurswith botulinum toxin), cognitive and systemic effects (which occur withcentral nervous depressants such as baclofen, tizanidine,benzodiazepines etc.).

The injections may be used to preserve and restore range of motionwithout surgery in case of subtle contractures for which there iscurrently no treatment other than stretching and serial casting whichcan cause prolonged discomfort and may be ineffective if not sustained.

An application of the invention is as a minimally-invasive localinjection at one or more CCs to treat limitation in movement and paindue to muscle stiffness, spasticity and mild contracture afterneurologic injury such as stroke, spinal cord injury, traumatic braininjury, cerebral palsy, and multiple sclerosis.

Another application is in the treatment of muscle stiffness due toprolonged immobility from medical conditions requiring prolonged bedrest or orthopedic conditions such as fractures, and surgery.

Another application is in the treatment of muscle and joint pain andstiffness from traumatic and post-surgery causes, myofascial pain,non-specific musculoskeletal pain, paratendonitis, periarthitis, ChronicRegional Pain Syndrome, fibromyalgia, frozen shoulder, nerve entrapmentand cheiroarthropathy (e.g. from diabetes).

Another application is to treat muscle contractures that occur due toreduced mobility in-utero or immediately after birth such asarthrogryposis multiplex congenita and brachial palsy from birth trauma.

Another application is to treat mild contractures and spasticityoccurring from rare diseases such as but not limited to amyotrophiclateral sclerosis, hereditary spastic paraplegia, mucopolysaccharidosis,spinal muscular atrophy, and Rett syndrome .

The present method may be used in any animal. In one embodiment, theindividual is a human being. In one embodiment, the individual is anon-human animal. It is generally considered that if the recipient is ahuman, the formulation will contain human hyaluronidase as the activeagent. Similarly, for a given animal, preferably, hyaluronidaseformulations comprising the active agent from the same species may beused. Based on the present disclosure locations of centers ofcoordination and/or center of fusion may be identified in animals foradministration of hyaluronidase. The dosage may be determinedempirically based on the site of the injection and extent of stiffnessor using a more objective measure of muscle stiffness such as anelastogram using ultrasonography. Determination of the dosage is withinthe purview of those skilled in the art. Although hyaluronidase is knownto be a short-acting drug (its activity is lost after about 12 hours),it was surprising that the patients treated with the present methodreported reduced muscle stiffness as observed by increased range ofmotion, ease of movement, and the presence of reduced number ofmovements that generate pain after 2 days which lasted for at leastabout 1 month. It was observed that any inflammation that results fromthe injections subsides in about 2 days.

In one aspect, this disclosure provides kits. The kits comprise one ormore of the following: one or more doses of hyaluronidase foradministration (either in ready-to-use form or in a form that needs tobe reconstituted), reconstitution medium (such as sterile saline,phosphate or other physiological buffers, water and the like),administration aids (such as syringes and the like), one or more chartsshowing centers for coordination and/or centers of fusion foradministration of hyaluronidase, and instructions for use. The chart mayidentify all the CCs and CFs or may identify CCs and CFs as being inspecific planes (sagittal, frontal and/or horizontal) in the front ofthe body and at the back of the body for restrictions in arm movement,leg movement, upper back problems, lower back problems and the like. Inone embodiment, the kit comprises one or more of the following: 2-20combined or individual doses of hyaluronidase, reconstitution medium forthe hyaluronidase, charts providing locations for centers forcoordination/centers of fusion in the body, and instructions for usesuch as instructions for reconstitution of the drug, audio and/or visualaids with instructions on assessing range-of-motion, selection ofCC-sites based on the restrictions noted on assessment of movement, aguide for amount to be injected based on the size of the individual,and/or instructions for an exercise program to be performed after theinjections are given. In one embodiment, the kit comprises 2-20 combinedor individual doses of from 25-300 units (per dose) of hyaluronidase anda chart (such as FIGS. 1A-1B and/or 2A-2B) providing centers ofcoordination or center of fusion points. In one embodiment, the kit maybe specific for a certain area. For example, the kit may contain from2-20 combined or individual doses of hyaluronidase, reconstitutionmedium, and a chart showing the CC and/or CF sites for a certain areasuch as the upper limb, shoulder upper back, neck and head, lower backand inferior limbs. In one embodiment, the individual doses are providedin ready to use syringes. The kits may be stored in the refrigerator orroom temperature (or any temperature therebetween).

EXAMPLE 1

This example describes the clinical use of a hyaluronidase formulationon 6 patients with chronic upper limb spasticity from a stroke (meantime since stroke was 5 years, range was 3-7 years). All the patientspresented with moderate spasticity as assessed with the ModifiedAshworth Scale (mean score was 3). Patients were selected because theyhad exhausted all current options.

The patients presented with lack of full pain-free range of motionpre-injection as evaluated by measuring their passive range of motion(Norkin and White, A Guide to Goniometry. Philadelphia, Pa.: FA DavisCo, 1995), from videos using Dartfish video analysis software (version3.0.2). The videos were taken in a standard manner with the cameraplaced perpendicular and at a distance of 1 meter from the jointevaluated. Illustrations of video images are shown in FIGS. 5A-5B, 6,7A-7C, 8A-8C, 9A-9B, 10A-10B, 11A-11B, and 12A-12F and results areprovided in table 1.

TABLE 1 Pre-injection Day 2 Follow-up Normal range of degrees degreesdegrees motion in degrees (SE) (SE) (SE) Shoulder Flexion 116 (7.57) 129(6.83) 137 (6.61) (movement of the arm forward) (0-180) Abduction 117(6.06) 145 (11.39) 155* (10.00) (raising the arm up to the side) (0-180)Elbow Flexion 127 (3.75) 133 (4.16) 128 (4.23) (bending the elbow)(0-160) Extension 171 (3.42) 172 (5.16) 174 (3.46) (straightening theelbow) (90-180) Pronation 85 (7.90) 94* (6.39) 97* (3.48) (forearmrotation to face palm down) (0-100) Supination 70 (12.39) 90* (8.31) 89*(13.04) (forearm rotation to face palm up) (0-90) Wrist Flexion 69(6.98) 70 (7.42) 61 (7.47) (bending the wrist down) (0-90) Extension 31(3.90) 56* (8.32) 46* (3.31) (bending the wrist up) (0-70) Ulnardeviation 40 (3.94) 44 (2.54) 44 (1.22) (pulls the hand toward themidline of the body) (0-65) Radial deviation 15 (5.19) 22 (4.40) 20(3.32) (pulls the hand away from the midline of the body) (0-25) Table1: Mean range of motion across 6 subjects pre-injection, 2 dayspost-injection and at approximately 4-6 week follow up. Statisticallysignificant differences compared to pre-injection values. Thecomparisons were made using Wilcoxon matched-pairs signed rank test.

The majority of the patients presented with limited passive range ofmotion pre-injection in the following joints: wrist extension (59% lessthan normal range of motion); ulnar deviation (43% less than normalrange of motion); shoulder abduction (37% less than normal range ofmotion); shoulder flexion (36% less than normal range of motion); elbowflexion (26% less than normal range of motion); wrist flexion (25% lessthan normal range of motion); and forearm supination (23% less thannormal range of motion).

Prior to the procedure a preliminary skin test for hypersensitivity toHylenex recombinant was performed. An intradermal injection ofapproximately 0.02 mL (3 Units) of a 150 Unit/mL solution was injected.No erythema, itching or wheal were noted at 5 or 20 minutes.

Dilution: 1:1 dilution with normal saline: 3 ml of hyaluronidase wscombined with 3 ml of normal saline. 6 injections of 1 ml each ofhylenex plus normal saline (Hylenex was 0.5 mls) were administered into6 separate fascial areas.

Location of Injections: the locations of the injections were selectedduring the clinical assessment as 6 fascial areas in the upper limb thatshowed the greatest stiffness and appeared to be limiting motion at thecorresponding joints in each patient. The six sites were selected fromthe following sites depending on individual presentation: an-sc, an-cu,re-cu, ir-cu, me-cu, la-cu, la-ca, it-ca, and re-ca.

Summary of Outcome: At two-day follow up, the patients reportedincreased range of motion in most of the joints targeted by theinjections. The increase in passive range of motion is shown in table 1.The patients who had active movement capability also showed an increasein the active range of motion.

The patients showed a statistically significant increase in range ofmotion for forearm pronation and supination and wrist extension 2 dayspost-injection, and for shoulder abduction, forearm pronation andsupination and wrist extension at follow-up. The comparisons were madeusing Wilcoxon matched-pairs signed rank test.

The movements that did not show an improvement were because there waslittle limitation in initial range of motion at these joints (table 1),and because we limited the location of the injections to 6 most impairedand painful areas for this cohort. An example of a patient treated withthis is provided in Example 2 below.

EXAMPLE 2

Problem: A 58 year old male patient sustained a stroke 2 years earlierand had severe pain and restriction of passive range of motion in hisright upper limb. The pain was a complaint and described subjectively bythe patient. Objectively, the patient could not tolerate passivemovements in the shoulder, elbow and wrist joints due to pain. Therewere no signs of joint or skin inflammation. There had been noimprovement in his neurologic or functional status despite the use oforal medications and Botulinum toxin injections.

Prior to the procedure a preliminary skin test for hypersensitivity toHYLENEX recombinant was performed. An intradermal injection ofapproximately 0.02 mL (3 Units) of a 150 Unit/mL solution was injected.No erythema, itching or wheal were noted at 5 or 20 minutes.

Dilution: 1:1 dilution with normal saline: 3 ml of hyaluronidase with 3ml of normal saline was prepared for administration.

Dedicated Physical Exam: Passive range of motion at the shoulder, elbow,forearm, wrist and fingers of the right upper limb were recorded. Thetone during passive range of motion was severe according to the ModifiedAshworth Scale (MAS) as indicated in table 2 below. The MAS is the mostcommon and frequently used measure of spasticity in adults and children,both in research and clinical practice (Bakheit et al 2003, J NeurolNeurosurg Psychiatry, May, 74(5):646-8. PubMed PMID: 12700310). MAS wasproposed by Bohannon and Smith in the 1987 and spasticity is rated on ascale from 0-4 as shown in Table 2.

TABLE 2 Modified Ashworth Scale rating score Grade Description 0 Noincrease in muscle tone 1 Slight increase in muscle tone, manifested bya catch and release or b minimal resistance at the end of the ROM whenthe affected pasr(s) is moved in flexion or in extention   1+ Slightincrease in muscle tone, manigested by a catch, followed by minimalresistance throughout the remainder (less than half) of ROM 2 Moremarked increase in muscle tone, throughout most of the ROM, but affectedpart(s) easily moved 3 Considerable increase in muscle tone, passivemovement is difficult 4 Affected part(s) rigid in flexion or extension

The right arm could be externally rotated at the shoulder but shoulderflexion and abduction were limited to 90 degrees and were extremelypainful. The right arm could be fully extended at the elbow. The wristcould be brought to neutral. However the forearm could not be supinatedand the wrist could not be extended past neutral due to subjectivereports of pain.

All injected areas were cleaned with chloroprep swabs prior toinjecting.

Units used were 150 units per ml.

Location of Injections: 6 injections of 1 ml each of hylenex plus normalsaline (hylenex was 0.5 mls) were injected into the areas described intable 3 at CCs an-sc, re-cu, an-ca, la-ca, re-ca, it-cu

TABLE 3 Modified Ashworth Volume Side score (cc) #Units SHOULDER In theclavi-coraco-axillary R 3 1 75 fascia, that unites the monoarticular(pectoralis minor) and biarticular (pectoralis major) muscle fibers(an-sc). UPPER ARM Over the fascia around the R 3 1 75 belly of tricepsat the level of deltoid's distal tendon (re-cu) LOWER ARM In theantebrachial fascia, R 3 1 75 in the point where the monoarticular(flexor carpi radialis) and biarticular (flexor pollicis longus) musclefibres unite (an-ca) In the fascia around the R 3 1 75 extensor carpiradialis (la-ca) Between the extensor R 3 1 75 digitorum and extensorpollicis longus (re-ca) In the fascia around the R 3 1 75 pronator teres(ir-cu) Table 3: Treatment protocol for patient 1.

Summary of Procedure: The patient tolerated the procedure well.Hemostasis was achieved. The patient left the office in stablecondition. Post-injection precautions were reviewed with the patient. Hewas advised to use warm compress for soreness related to injections. Hewas told to resume normal activity and the caregiver was advised tostretch his arm frequently.

Summary of Outcome: The injections had a dramatic effect on the patienton day 2. He was able to tolerate shoulder abduction to 90° for aprolonged period. Previously we were unable to achieve that degree ofmotion secondary to extreme pain reported subjectively. His therapistcreated a simple positioning device for him to maintain the position atthe shoulder and provide a slow stretch. The patient's spasticitydecreased from Ashworth score of 3 to 1 at all the joints in two days.

These data demonstrate the effectiveness of the present method.

EXAMPLE 3

This example provides further illustration of this method.

Problem: A fifteen year old boy sustained a basketball injury to theneck which led to a stroke 8 months ago with severe muscle stiffness andlack of passive and active range of motion in his right upper limb. Hereceived hyaluronidase injections for the first time 3 months which ledto dramatic improvement in shoulder range of motion both passively andactively. However the wrist and hand were not addressed at that time.Objectively, the patient kept the fingers of his right hand curled andmaintained the wrist in a flexed position. Passive movements werepainful as they needed to be forced. There were no signs of joint orskin inflammation. Other modalities including oral medication,therapeutic exercise and stretching did not relieve the tightness in thewrist and hand.

Prior to the procedure a preliminary skin test for hypersensitivity toHYLENEX recombinant was performed. An intradermal injection ofapproximately 0.02 mL (3 Units) of a 150 Unit/mL solution was injected.No erythema, itching or wheal were noted at 5 or 20 minutes.

Dilution: 1:1 dilution with normal saline: 4 ml of hyaluronidase with 4ml of normal saline was prepared for administration.

Dedicated Physical Exam: Active and passive range of motion at theshoulder, elbow, forearm, wrist and fingers of the right upper limb wererecorded. The tone during passive range of motion was severe accordingto the Modified Ashworth Scale (MAS) as indicated in table 4 below.

Full active range-of-motion at the shoulder and elbow and forearmsupination were recorded. Active range-of-motion was restricted forforearm pronation, wrist extension and finger extension. Specifically,range of motion for pronation was 90-45 degrees, there was no activemotion at the wrist and the fingers could not be extended fully at theproximal inter-phalangeal joints passively or actively.

All injected areas were cleaned with chloroprep swabs prior toinjecting.

Units used were 150 units per ml.

Location of Injections: 8 injections of 1 ml each of hylenex+normalsaline were injected into the areas described in table 4 at CCs la-cu,it-ca, an-ca, me-cu, re-ca, it-cu, re-di, er-di, it-di.

TABLE 4 Volume Side Ashworth (cc) #Units LOWER ARM In the fascia aroundthe R 2 1 75 brachioradialis (la-cu) In the fascia around the flexor R 31 75 digitorum (ir-ca) In the fascia around the flexor R 3 1 75 carpiradialis (an-ca) In the fascia around the flexor R 3 1 75 carpi ulnaris(me-cu) Between the extensor carpi ulnaris R 3 1 75 and the extensordigitorum (re-ca) In the fascia around the pronator R 2 1 75 teres(ir-cu) HAND AND WRIST In the dorsal aspect of the hand R 3 1 75 betweenthe metacarpal bones (re-di, er-di); 2 sites In the palmar fascia R 3 175 (ir-di) Table 4: Treatment protocol for patient 2.

Summary of Procedure: The patient tolerated the procedure well.Hemostasis was achieved. The patient left the office in stablecondition. Post-injection precautions were reviewed with the patient. Hewas advised to use warm compress for soreness related to injections. Hewas told to resume normal activity and given a home exercise program tostretch the forearm, wrist and fingers.

Summary of Outcome: The injections had a dramatic effect on the patientexamined 1 week later. Forearm pronation was observed to have fullactive pronation with some shoulder strategy; previously 90 to 45degrees. Wrist extension was from 80 degrees of flexion to slightly pastneutral. Wrist flexion was 0-80 passive, some active with gravityassist. Finger flexion-extension (MCP) was 90-0 degrees with PIP'sextended (MAS=1). He could keep the fingers extended at rest and foldthe hands together in praying position. The Ashworth score decreasedfrom 3 to 1 for the greatest areas of muscle stiffness.

These data demonstrate the effectiveness of the present method.

While the method has been described through specific embodiments,routine modifications will be apparent to those skilled in the art,which modifications are intended to be within the scope of thedisclosure.

The invention claimed is:
 1. A method for providing relief fromstiffness of a first muscle in an individual comprising the steps ofdelivering in a region of deep fascia surrounding the first muscle or ina region of deep fascia surrounding a second muscle that affects thefunction of the first muscle at or near one or more centers ofcoordination or center of fusion associated with the first muscle,second muscle or a combination thereof, a composition comprising atherapeutically effective amount of hyaluronidase, wherein a center ofcoordination or center of fusion is characterized by slidable layers inthe deep fascia, and wherein delivering the composition results inreduction of stiffness of the first muscle.
 2. The method of claim 1,wherein hyaluronidase is administered via injections at multiple centersof coordination.
 3. The method of claim 2, wherein the amount ofhyaluronidase for each injection is about 25 to 300 USP units.
 4. Themethod of claim 2, wherein the hyaluronidase injections are carried outat 2-6 CC locations.
 5. The method of claim 2, wherein hyaluronidaseinjections are carried out at 6-10 CC locations.
 6. The method of claim2, wherein the hyaluronidase injections are repeated over a period ofbetween 1-12 months.